Tissue factor pathway inhibitor (TFPI) rapidly inhibits both factor Xa and the factor VIIa/tissue factor catalytic complex. Thus, it is thought to be the most important inhibitor of the initiation of blood coagulation. Although its structure suggests that it is a soluble protein, most TFPI is associated with the vascular endothelium.The objective of this proposal is to define the biochemical mechanisms responsible for the association of TFPI with biological surfaces. Heparin infusion results in a prompt 2-to 10-fold increase in circulating TFPI concentration, therefore, interactions with glycosaminoglycans are considered a primary mode of cell surface association. However, flow cytometry studies demonstrate that over 95 percent of the TFPI on the surface of cultured endothelial cells is bound through a glycosyiphosphatidylinositol (GPI)-anchor in a manner that is not altered by heparin. We have previously shown that glypican-3, a GPI-anchored proteoglycan, binds to TFPI and may account for TFPI binding to the endothelium. Recently, we identified thrombospondin - 1 (TSP) as a second TFPI binding protein that may act to recruit and localize TFPI to extravascular surfaces within a bleeding wound. This revised proposal is organized into three specific aims that reflect the three long term approaches our laboratory is taking to investigate the function of TFPI as a surface associated inhibitor of tissue factor initiated blood coagulation.Specific Aim #1 is focused on in vitro structure/function studies of TFPI. Proposed projects include: the investigation of the role of the interaction between TFPI and TSP in the cellular catabolism of TFPI; defining the mechanisms responsible for the proliferative effect of TFPI on endothelial cells; and enzyme kinetic studies with altered forms of TFPI to further define the role of the third Kunitz domain and C-terminal region in the anticoagulant activity of TFPI.Specific Aim #2 involves in vivo/ex vivo studies using human placenta and TSP knock-out mice to define the relative amounts of GPI-anchored and heparin-releasable TFPI found within vascular beds in vivo and to characterize their structures. The studies with the TSP knock-out mice will define the role of TSP in the association of TFPI with the endothelial surface.Specific Aim #3 is designed to characterize how TFPI is processed within the cell to produce the heparin-releasable and GPI-anchored pools. Recombinant TFPI lacking the first two, non-translated, exons appears to be entirely secreted into the culture medium. Possible explanations for this observation include that the first two exons are required for proper intracellular targeting to a GPI-anchored binding protein or that the GPI-anchored binding sites are saturated. These hypotheses will be tested using TFPI constructs containing the first two exons. Additionally, TFPI constructs that have been tagged either with the HA or the FLAG tag will be transfected into cells to allow differentiation of endogenous and recombinant surface TFPI in flow cytometry experiments. Pulse-chase immunoprecipitation studies will be performed using Trition-X-1 14 for cell lysis. These experiments will define how long after synthesis and the percentage of the total TFPI that becomes attached to a GPI-anchor.These proposed studies will provide a detailed characterization of how TFPI associates with the vascular endothelium and establish models for the study of TFPI within different vascular beds. These studies are highly clinically relevant because TFPI is a an important inhibitor of tissue factor initiated blood coagulation that produces the pathological thrombi in stroke, heart attack and many other thrombotic diseases.